Process for producing erythromycin



2,809,151 PROCESS FOR PRODUCING ERYTHROMYCIN Le Roy E. Johnson, Kalamazoo, Mich., assignor to The Upjohn Company, Kalamazoo, Mich., a corporation of Michigan No Drawing. Application June 12, 1953, Serial No. 361,417

Claims. (Cl. 195-580.)

This invention relates to the fermentation culturing of erythromycin ferment and is particularly directed to methods and compositions for obtaining improved yields of erythromycin characterized by the incorporating in the erythromcyin substrate medium of a water-soluble salt of propionic acid.

It is an object of the invention to provide new and improved processes for producing erythromycin. It is another object of the invention to provide new and improved substrate media for culturing erythromycin ferment; It is another object of the invention to provide adjuvants which when added to erythromycin substrate media, function to increase the yield of erythromycin. Still other objects will appear as the description proceeds.

These objects are accomplished in the present invention by incorporating in the erythromycin substrate medium used in culturing the erythromycin ferment a watersoluble salt of propionic acid in an amount suflicient to stimulate the production of erythromycin. Through the addition of water-soluble salts of propionic acid to erythromycin substrate and culturing erythromycin ferment therein, a yield of erythromycin is obtained which is substantially greater than the yield obtainable in the absence of water-soluble salts of propionic acid.

In carrying out the processes of the invention, an erythromycin ferment such as Streptomyces erythreus NRRL 2338 is cultured in an erythromycin substrate medium according to the invention using procedures and apparatus well understood in the art. See U. Patent 2,653,899, and Antibiotics and Chemotherapy 2: 281-283 (June 1952). The fermentation is carried out under aerobic conditions and advantageously by the submerged culture process. Initially the substrate medium is adjusted to between pH 6.0 and pH 7.5, advantageously to about pH 6.5. In the fermentation the pH ordinarily increases up to about pH 9. Suitably the temperature may range between about 25 degrees centigrade and about 37 degrees centigrade, advantageously between 26 and 32 degrees centigrade. In these and other respects the procedure outlined in the above U. S. Patent 2,653,899 can be advantageously followed.

The basic erythromycin substrate is carbohydrate. Preferred sources of carbohydrate are starch and dextrose (glucose). Other sources which can be included or substituted for starch and dextrose are sucrose, dextrin, molasses, brown sugar, maltose, and the like. The erythromycin substrate media advantageously contain in addition, nitrogen sources, preferably such organic nitrogen sources as corn steep, soy bean meal or flour, distillers solubles, casein, amino acid mixtures, peptones (both meat and soy), brewers yeast, peanut granules, peanut meal, and the like. Inorganic nitrogen sources such as nitrate salts or ammonium salts can also be employed. The substrate media also can contain nutrientinorganic salts which furnish sodium, potassium, calcium, phosphate, chloride, sulfate, and like ions. Essential trace elements such as cobalt, magnesium, iron, and manganese can be Patented Oct. 8, 1957 "ice added though sufiicient amounts of such elements are ordinarily supplied by the other constituents of the substrate medium.

A suitable basic erythromycin substrate medium advantageously contains from about 0.5 to about ten percent carbohydrate sources, from about 0.5 to about five percent nitrogen sources, up to about one percent nutrient salts, up to about 0.5 percent calcium carbonate or like buifer salt, and the balance substantially all water. To this basic erythromycin substrate medium there is added in accordance with the invention a water-soluble salt of propionic acid in an amount suiiicient to stimulate the production of erythromycin. Ordinarily the amount of material will not exceed about one percent. Any smaller amount down to the minimal effective concentration can be used. Ordinarily it is not desirable to use less than about 0.1 percent though effective stimulation has been observed at lower concentrations down to about 0.05 percent. (The parts and percentages used herein are by weight unless otherwise specified.)

The adjuvant material according to the invention advantageously is added to the erythromycin substrate medium prior to or at the time of inoculation. It can be added before the substrate medium is sterilized or sterile material can be added thereafter but prior to inoculation. The desired quantity of said adjuvant can be added all at the beginning or at periodic intervals during the fermentation. In the latter case, the adjuvant can be added in an aggregate amount greater than one percent. The material can be incorporated by forming a salt in situ, for example, by adding propionic acid and readjusting the pH. In such case, the propionic acid salt of the base used to adjust the pH is formed.

Suitable adjuvants according to the invention which can be added to basic erythromycin substrate media to increase erythromycin'production, include sodium propionate and other alkali-metal (including ammonium) salts and alkaline earth-metal salts (including magnesium), or in other words, the light metal salts. The heavy metal propionic acid salts of non-toxic heavy metals such as iron, cobalt, and manganese can also be used.

The invention can now be more fully understood by the following examples which are illustrative of the processes and composition of the present invention, but are not to be construed as limiting.

EXAMPLE 1 A sporulated culture is produced by growing Streptomyces erythreus strain NRRL 2338 on a nutrient agar slant. The spores are recovered in water suspension and used to inoculate a substrate medium suitable for producing a vegetative growth. The spore suspension is introduced into a SOO-mil flask containing mils of the following sterile substrate medium:

Dextrose (technical grade) grams 10 Beef extract do 10 Sodium chloride, -do 5 Bacto-peptone do 5 Water mils 1000 and incubated at 28.5 degrees centigrade for three days on a reciprocal shaker.

following erythromycin substrate medium in the proportions indicated in the table:

Technical dextrose grams 25 Soya bean meal (partially defatted) do 25 Brewers yeast (debittered) do 5 Sodium chloride d Calcium carbonate do 2 Water mils 1000 Table I.Efiect of various adjuvams on yield of erythromycin Concen- Adjuvant tration Assay, Mierograms/mil in percent FIRST SERIES 5 day 6 day 7 day None 0 200 263 184 Sodium propionate 0. 4 403 117 349 Propionic acid 1 0. 4 293 455 313 SECOND SERIES None 0 125 148 145 Sodium propionate. 0. 4 405 533 555 Calcium propionate 0. 4 210 373 343 1 The pH was readjusted to 6.6 by addition of sodium hydroxide solution. In all other cases initial pH was about 6.6.

In the following table there is illustrated the effect of the concentration of adjuvant upon the yield of erythromycin:

Table II.Efiect of various amounts of sodium propio- In the second series the dcfatted soy bean meal of the first series was substituted by whole soy bean meal.

EXAMPLE 2 Following the technique detailed in Example 1, an

erythromycin substrate medium having the following composition:

Dextrose grams 30 Peanut granules do 25 Brewers yeast (debittered) do 5 Sodium chloride do 5 Calcium carbonate do.. 2 Cellulose do 10 Water mils 1000 was inoculated and cultured at 32 F. with aeration at the rate of six liters of air per mintue in eight-liter batches in five-gallon bottles provided with sweep-stirrers. As in Example 1, aseptic conditions were maintained throughout. The peanut granules were whole raw peanuts granulated to an average particle size in the order of one-eighth to one-quarter of an inch. The results obtained are set forth in the following table:

Table III.Efiect of 0.2 percent sodium propionate in sweepstir bottles with peanut granules as a substrate.

It is to be understood that the invention is not limited to the exact details of operation or exact compounds shown and described as obvious modifications and equivalents will be apparent to one skilled in the art and the invention is therefore-to be limited only by the scope of the appended claims.

I claim:

1. In the manufacture of erythromycin by the fermentation culturing of Streptomyces erythreus, the method which comprises culturing Streptomyces erythreus in an erythromycin substrate medium containing about 0.05 to about one percent of a water-soluble salt of propionic acid.

2. In the manufacture of erythromycin by the fermentation culturing of Streptomyces erythreus in a substrate medium containing assimilable carbohydrate, protein, and minerals, the improvement which comprises adding to said substrate medium a stimulating amount greater than about 0.05 percent of a water-soluble salt of propionic acid.

3. In the manufacture of erythromycin by the fermentation culturing of Streptomyces erythreus, the method which comprises culturing Streptomyces erythreus in an erythromycin substrate medium containing about 0.05 to about one percent of a light metal propionate.

4. In the manufacture of erythromycin by the fermentation culturing of Streptomyces erythreus, the method which comprises culturing Streptomyces erythreus in an erythromycin substrate medium containing about 0.05 to about one percent of sodium propionate.

5. In the manufacture of erythromycin by the fermentation culturing of Streptomyces erythreus, the method which comprises culturing Streptomyces erythreus in an erythromycin substrate medium containing about 0.05 to about one percent of calcium propionate.

Levine et al.: Compilation of Culture Media, William & Wilkins, 1930, pages 5 and 31.

Porter: Bacterial Chemistry and Physiology, Wiley. 1946, pages 800-802. 

1. IN THE MANUFACTURE OF ERYTHROMYCIN BY THE FERMENTATION CULTURING STREPTOMYCES ERYTHREUS, THE METHOD WHICH COMPRISES CULTURING STREPTOMYCES ERYTHREUS IN AN ERYTHROMYCIN SUBSTRATE MEDIUM CONTAINING ABOUT 0.05 TO ABOUT ONE PERCENT OF A WATER-SOLUBLE SALT OF PROPIONIC ACID. 